Recombinant DNA I
Basics of molecular cloning
Polymerase chain reaction
cDNA clones and screening
Recombinant DNA Technology
• Utilizes microbiological selection and screening
procedures to isolate a gene that represents as
little as 1 part in a million of the genetic material in
an organism.
• DNA from the organism of interest is divided into
small pieces that are then placed into individual
cells (usually bacterial).
• These can then be separated as individual
colonies on plates, and they can be screened to
find the gene of interest.
• This process is also called molecular cloning.
DNA pieces are joined in vitro to form
recombinant molecules
• Generate sticky ends on the DNA, e.g. with
restriction endonucleases
• Tie DNA molecules from different sources
together with DNA ligase
Restriction endonucleases generate ends
that facilitate mixing and matching
GAATTC
CTTAAG
GAATTC
CTTAAG
EcoRI cut
G
AATTC
CTTAA
G
G
AATTC
CTTAA
G
Mix and ligate
G AATTC
CTTAA G
Recombinant
molecules
G AATTC
CTTAA G
GAATTC
CTTAAG
GAATTC
CTTAAG
Parental
molecules
DNA ligase covalently joins two DNA molecules
• UsesDNA
ATP
or NADH to provide energy to seal nicks
ligase will seal the nicks that remain after annealing two fragments together
nick
P
P
P
A
T
OH
P
G
C
P
G
C
P
P
A
T
P
P
A
T
P
P
T
A
P
P
T
A
P
C
G
P
G
C
P
T
A
P
P
OH
P
P
A
T
P
P
nick
T4 DNA ligase + ATP
P
P
A
T
G
C
P
P
P
G
C
P
P
A
T
P
P
A
T
P
P
T
A
P
P
T
A
P
P
C
G
P
P
G
C
P
P
T
A
P
A
T
P
P
Alternate method to join DNA:
homopolymer tails
Alternate
method to
join DNA:
linkers
Introduction of recombinant DNA into
living cells via vectors
• Autonomously replicating DNA molecules
– (have an origin of replication)
• Selectable marker, such as drug resistance
• Insertion site for foreign DNA
– (often a genetically engineered multiple cloning
region with sites for several restriction enzymes)
Plasmid vectors
• Circular, extrachromosomal, autonomously
replicating DNA molecules
• Frequently carry drug resistance genes
• Can be present in MANY copies in the cell
A common plasmid cloning vector: pUC
lacZ
mulitple
cloning
sites
pUC
ApR
ColE1 origin
of replication
Lac+, or blue colonies
on X-gal in
appropriate
strains of E. coli
High copy
number
foreign DNA
lacZ
pUC recombinant
ApR
ColE1 ori
Lac-, or white colonies
on X-gal in
appropriate
strains of E. coli
Transformation of E. coli
• E. coli does NOT have a natural system to
take up DNA
• Treat with inorganic salts to destabilize cell
wall and cell membrane
• During a brief heat shock, some of the
bacteria takes up a plasmid molecule
• Can also use electroporation
Phage vectors
• More efficient introduction of DNA into
bacteria
• Lambda phage and P1 phage can carry
large fragments of DNA
– 20 kb for lambda
– 70 to 300 kb for P1
• M13 phage vectors can be used to generate
single-stranded DNA
YAC vectors for cloning large DNA inserts
ori
TRP1
Yeast artificial chromosome = YAC
CEN4 SUP4
S
pYAC3
TEL
TEL
B B
11.4 kb
URA3
Cut with restriction
Enzymes S + B
Ligate to very large
Fragments of genomic
DNA
TEL TRP1 ori CEN4
URA3 TEL
Large insert, 400 to
as much as 1400 kb
Not to scale.
Bacterial artificial chromosomes
• Are derived from the fertility factor, or Ffactor, of E. coli
• Can carry large inserts of foreign DNA, up to
300 kb
• Are low-copy number plasmids
• Are less prone to insert instability than YACs
• Have fewer chimeric inserts (more than one
DNA fragment) than YACs
• Extensively used in genome projects
BAC vectors for large DNA inserts
Cm(R)
oriF
promoter
S
E
E
pBACe3.6
11.5 kb
SacBII
SacB+: SacBII encodes levansucrase,
which converts sucrose to levan,
a compound toxic to the bacteria.
Cut with restriction enzyme E, remove “stuffer”
Ligate to very large fragments of genomic DNA
promoter
S
Cm(R)
Not to scale.
Large insert, 300kb
oriF
SacBII
SacB-: No toxic levan produced on sucrose
media: positive selection for recombinants.
PCR provides access to specific DNA segments
• Polymerase Chain Reaction
• Requires knowledge of the DNA sequence
in the region of interest.
• As more sequence information becomes
available, the uses of PCR expand.
• With appropriate primers, one can amplify
the desired region from even miniscule
amounts of DNA.
• Not limited by the distribution of restriction
endonuclease cleavage sites.
Polymerase chain reaction, cycle 1
Primer 1
Primer 2
Template
Cycle 1 1. Denature
2. Anneal primers
3. Synthesize new DNA with polymerase
Polymerase chain reaction, cycle 2
Cycle 2 1. Denature
2. Anneal primers
3. Synthesize new DNA with polymerase
PCR, cycle 3
Cycle 3 (focus on DNA segments bounded by primers)
1. Denature
2. Anneal primers
3. Synthesize new DNA with polymerase
2 duplex
molecules
of desired
product
PCR, cycle 4: exponential increase in
product
Cycle 4: Denature, anneal primers, and synthesize new DNA:
6 duplex
molecules
of desired
product
PCR, cycle 5: exponential increase in
product
Cycle 5: Denature, anneal primers, and synthesize new DNA:
14 duplex
molecules
of desired
product
PCR: make large amounts of a
particular sequence
• The number of molecules of the DNA
fragment between the primers increases
about 2-fold with each cycle.
• For n = number of cycles, the amplification
is approximately [2exp(n-1)]-2.
• After 21 cycles, the fragment has been
amplified about a million-fold.
• E.g. a sample with 0.1 pg of the target
fragment can be amplified to 0.1 microgram
PCR is one of the most widely used
molecular tools in biology
• Molecular genetics - obtain a specific DNA
fragment
– Test for function, expression, structure, etc.
• Enzymology - place fragment encoding a
particular region of a protein in an expression
vector
• Population genetics - examine polymorphisms in a
population
• Forensics - test whether suspect’s DNA matches
DNA extracted from evidence at crime scene
• Etc, etc